PROJECT SUMMARY/ABSTRACT A fundamental question is whether physiological variations in metabolite levels in vivo influence stem cell function or tissue regeneration. Our understanding of somatic stem cell metabolism remains limited due to the technical challenges associated with studying metabolism in rare cells in vivo. To address this issue we optimized the sensitivity of metabolomics methods to enable the analysis of rare cell populations. We used this approach to compare metabolite levels between hematopoietic stem cells (HSCs) and a wide range of restricted hematopoietic progenitors isolated from mouse bone marrow. We found that each hematopoietic stem and progenitor cell population had a distinct metabolic signature. Human and mouse HSCs were distinguished from other hematopoietic cells by unusually high levels of ascorbate (vitamin C). Ascorbate depletion in mice increased HSC frequency and function, partly by reducing the activity of Tet2, a cytosine demethylase that suppresses leukemia development. Ascorbate depletion, like Tet2 deletion, cooperated with Flt3ITD to promote myelopoiesis and leukemogenesis. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis mainly through Tet2-dependent mechanisms (see schematic in Figure 1). These observations are likely relevant to public health as Americans exhibit a 10-fold variation in plasma ascorbate levels, largely due to dietary differences. At any one time, approximately 5% of Americans have ascorbate levels that are comparable to the ascorbate-depleted mice we studied. In Aim 1, we propose to test whether ascorbate levels influence steady-state hematopoiesis or regeneration after hematopoietic stresses. In Aim 2, we propose to test whether ascorbate depletion promotes clonal hematopoiesis under steady state conditions or in response to hematopoietic stresses. Clonal hematopoiesis of indeterminate potential has recently been shown to be common in healthy older people as well as in patients with aplastic anemia, solid cancers, and patients who have received hematopoietic transplants. The presence of clonal hematopoiesis is associated with adverse health outcomes including cardiovascular disease. Clonal hematopoiesis is usually caused by the loss of one allele of Tet2 or by loss- of-function mutations in Dnmt3a. We hypothesize that ascorbate depletion can promote the development and progression of clonal hematopoiesis by reducing Tet2 function. In Aim 3 we will assess the molecular mechanisms by which ascorbate depletion and Tet2 deficiency regulate HSC function and myelopoiesis. We expect these studies to expand our understanding of how metabolism regulates the HSC epigenome, HSC function, normal hematopoiesis, and clonal hematopoiesis.